US7056687B2ExpiredUtilityPatentIndex 70

Methods and compositions for screening for altered cellular phenotypes

Assignee: RIGEL PHARMACEUTICALS INCPriority: May 12, 1998Filed: Mar 8, 2002Granted: Jun 6, 2006

Est. expiryMay 12, 2018(expired)· nominal 20-yr term from priority

Inventors:LORENS JAMES KINSELLA TODD M MASUDA ESTEBAN HITOSHI YASUMICHI LIAO X CHARLENE PEARSALL DENISE FREIRO ANNABELLE CHU PETER

C12N 15/1079G01N 33/5047C12N 15/63G01N 33/5008C07K 14/70578C07K 2319/00G01N 33/5023C12N 2510/00C12Q 1/6897G01N 2500/10

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Claims

Abstract

The invention relates to methods and compositions useful for screening for altered cellular phenotypes using an inducible expression system to enrich for and detect the altered phenotypes and, more particularly, relates to screening libraries of candidate bioactive agents, for example, nucleic acids and peptides, in cells using an regulatable expression system to enrich for a subpopulation of cells having an altered phenotype due to the presence of a candidate bioactive agent.

Claims

exact text as granted — not AI-modified

We claim:

1. A method of screening for cells having an altered phenotype, the method comprising the steps of:
a) providing a population of cells having a parent phenotype, said population of cells comprising a nucleic acid sequence encoding a first element expressed in said population of cells;
b) introducing into said population of cells a library of fusion nucleic acids, said fusion nucleic acids each comprising:
(1) a second element that is regulatable by said first element; and
(2) a nucleic acid sequence encoding a candidate bioactive agent, wherein said nucleic acid sequence is operably linked to said second element;

c) modulating the expression of said nucleic acid sequence encoding the candidate bioactive agent by contacting said population of cells with a third element, wherein said modulation is selected from the group consisting of induction and repression of said nucleic acid sequence encoding the candidate bioactive agent;
d) collecting a first subpopulation of cells having an altered phenotype;
e) modulating in the opposite direction of step (c) the expression of said nucleic acid sequence by modulating said contacting of first subpopulation of cells with said third element;
f) collecting a second subpopulation of cells having said parent phenotype;
g) modulating in the same direction as in step (c) the expression of said nucleic acid sequence by contacting said second subpopulation of cells with said third element; and
h) detecting a third subpopulation of cells having said altered phenotype.

2. The method according to claim 1 further comprising:
i) collecting said third subpopulation of cells having said altered phenotype;
j) modulating in the opposite direction of step (c) the expression of said nucleic acid sequence by modulating said contacting of said third subpopulation of cells with said third element; and k) detecting a fourth subpopulation of cells having said parent phenotype.

3. The method according to claim 2 further comprising:
I) collecting said fourth subpopulation of cells having said parent phenotype;
m) modulating in the same direction as in step (c) the expression of said nucleic acid sequence by contacting said fourth subpopulation of cells with said third element; and
n) detecting a fifth subpopulation of cells having said altered phenotype.

4. A method of screening for cells having an altered phenotype, the method comprising the steps of:
a) providing a population of cells having a parent phenotype, said population of cells comprising a nucleic acid sequence encoding a first element expressed in said population of cells;
b) introducing into said population of cells a library of fusion nucleic acids, said fusion nucleic acids each comprising:
1) a second element that is regulatable by said first element; and
2) a nucleic acid sequence encoding a candidate bioactive agent, wherein said nucleic acid sequence is operably linked to said second element;

c) inducing the expression of said nucleic acid sequence encoding the candidate bioactive agent by contacting said population of cells with a third element;
d) collecting a first subpopulation of cells having an altered phenotype;
e) repressing the expression of said nucleic acid sequence by modulating said contacting of first subpopulation of cells with said third element;
f) collecting a second subpopulation of cells having said parent phenotype;
g) inducing the expression of said nucleic acid sequence by contacting said second subpopulation of cells with said third element; and
h) detecting a third subpopulation of cells having said altered phenotype.

5. The method according to claim 4 further comprising:
i) collecting said third subpopulation of cells having said altered phenotype;
j) repressing the expression of said nucleic acid sequence by modulating said contacting of said third subpopulation of cells with said third element; and
k) detecting a fourth subpopulation of cells having said parent phenotype.

6. A method of screening for cells having an altered phenotype, the method comprising the steps of:
a) providing a population of cells having a parent phenotype, said population of cells comprising a nucleic acid sequence encoding a first element;
b) introducing into said population of cells a library of fusion nucleic acids, said fusion nucleic acids each comprising:
(1) a second element that is regulatable by said first element; and
(2) a nucleic acid sequence encoding a candidate bioactive agent, wherein said nucleic acid sequence is operably linked to said second element;

c) inducing the expression of said nucleic acid sequence encoding the candidate bioactive agent by expressing said first element in said population of cells;
d) collecting a first subpopulation of cells having an altered phenotype;
e) repressing the expression of said nucleic acid sequence by contacting said first subpopulation of cells with a third element;
f) collecting a second subpopulation of cells having said parent phenotype;
g)inducing the expression of said nucleic acid sequence by modulating said contacting of said second subpopulation of cells with said third element; and
h) detecting a third subpopulation of cells having said altered phenotype.

7. The method according to claim 6 further comprising:
i) collecting said third subpopulation of cells having said altered phenotype;
j) repressing the expression of said nucleic acid sequence by contacting said third subpopulation of cells with said third element; and
k) detecting a fourth subpopulation of cells having said parent phenotype.

8. The method according to claim 7 further comprising:
l) collecting said fourth subpopulation of cells having said parent phenotype;
m) inducing the expression of said nucleic acid sequence by modulating said contacting said fourth subpopulation of cells with said third element; and n) detecting a fifth subpopulation of cells having said altered phenotype.

9. The method according to any one of claims 4 – 5 , wherein said first element comprises a reverse tetracycline-dependent transactivator (rtTA).

10. The method according to any one of claims 6 – 8 , wherein said first element comprises a tetracycline-dependent transactivator (tTA).

11. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said second element comprises an tetracycline operator sequence (TetO).

12. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said second element comprises an oligomer of a tetracycline operator sequence (TetO).

13. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said third element comprises tetracycline (Tet).

14. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said third element comprises a tetracycline analogue.

15. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said third element comprises doxycycline (Tet).

16. The method according to any one of claims 4 – 5 , wherein:
said first element comprises a reverse tetracycline-dependent activator (rtTA);
said second element comprises an oligomer of a tetracycline operator sequence (TetO); and
said third element comprises tetracycline or doxycycline.

17. The method according to any one of claims 6 – 8 , wherein:
said first element comprises a tetracycline-dependent activator (rtTA);
said second element comprises an oligomer of a tetracycline operator sequence (TetO); and
said third element comprises tetracycline (Tet) or doxycycline (Dox).

18. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said population of cells comprise a stimulator and said parent phenotype is due to the presence of said stimulator.

19. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acids are each a component of a retroviral vector.

20. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said candidate bioactive agent is a polypeptide.

21. The method according to any-one of claims 4 – 5 and 6 – 8 , wherein said candidate bioactive agent is a cyclic polypeptide.

22. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said first element is expressed stably or transiently.

23. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said first element is expressed constitutively.

24. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said first element is expressed in trans or in cis relative to said candidate bioactive agent.

25. The method according to any one of claims 4 – 5 and 6 – 8 , wherein the expression of said first element is inducible.

26. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acids comprise said nucleic acid encoding said first element.

27. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said candidate bioactive agent is an RNA.

28. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said candidate bioactive agent is an antisense RNA.

29. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said candidate bioactive agent is a DNA.

30. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent comprises a full-length cDNA.

31. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent comprises a subsequence of a full-length cDNA.

32. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent comprises an antisense sequence of a full-length cDNA.

33. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent comprises an antisense sequence that is a subsequence of a full-length cDNA.

34. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encodes an amino acid sequence that is in-frame or out-of-frame as compared to the open reading frame (ORF)encoded by the amino acid sequence of a full-length cDNA, said amino acid sequence encoding said candidate bioactive agent.

35. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said library of fusion nucleic acids comprises about 10 3 to 10 9 different said nucleic acid sequences.

36. The method according to any one of claims 1 – 3 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent is a random nucleic acid sequence.

37. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said nucleic acid sequence encoding the candidate bioactive agent is a biased random nucleic acid sequence.

38. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said library comprises about 10 4 to 10 8 random nucleic acid sequences.

39. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a sequence encoding a reporter protein, wherein said reporter protein is operably linked to said nucleic acid sequence.

40. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a sequence encoding a reporter protein that is an autofluorescentprotein, wherein said reporter protein is operably linked to said nucleic acid sequence.

41. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a sequence encoding a reporter protein that is green fluorescent protein (GFP), wherein said reporter protein is operably linked to said nucleic acid sequence.

42. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a sequence encoding a reporter protein that is green fluorescent protein (GFP) from Aqueorea, wherein said reporter protein is operably linked to said nucleic acid sequence.

43. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a sequence encoding a reporter protein that is green fluorescent protein (GFP) from a Renilla species, wherein said reporter protein is operably linked to said nucleic acid sequence.

44. The method according to any of claims 4 – 5 and 6 – 8 , wherein said collecting is by fluorescence-activated cell sorting (FACS).

45. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said fusion nucleic acid further comprises a third nucleic acid sequence encoding a reporter protein that is green fluorescent protein (GFP) from a Renilla species, wherein said reporter protein is operably linked to said third nucleic acid sequence; and wherein said collecting is by fluorescence-activated cell sorting (FACS).

46. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said cells of said population are mammalian cells.

47. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of cell cycle regulation due to the presence of said candidate bioactive agent.

48. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of exocytosis due to the presence of said candidate bioactive agent.

49. The method according to any of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of T cell activation due to the presence of said candidate bioactive agent.

50. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of IgE synthesis due to the presence of said candidate bioactive agent.

51. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of IgE secretion due to the presence of said candidate bioactive agent.

52. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of antigen-induced B cell differentiation due to the presence of said candidate agent.

53. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of antigen-induced B cell isotype switching due the presence of said candidate agent.

54. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of IgE switching due to the presence of said candidate bioactive agent.

55. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of apoptosis due to the presence of said candidate bioactive agent.

56. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of angiogenesis due to the presence of said candidate bioactive agent.

57. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of T cell receptor (TCR) activation due to the presence of said candidate bioactive agent.

58. The method according to any one of claims 4 – 5 and 6 – 8 , wherein said altered phenotype comprises the modulation of a T cell surface marker due to the presence of said candidate bioactive agent, wherein said marker is selected from a group of markers consisting of CD3, CD25, CD28, CD40L, CD69, CD95, and CD95L.

59. The method according to claim 5 further comprising:
k) collecting said fourth subpopulation of cells having said parent phenotype;
l) the expression of said nucleic acid sequence by contacting said fourth subpopulation of cells with said third element; and
m) detecting a fifth subpopulation of cells having said altered phenotype.

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